U.S. patent application number 10/542883 was filed with the patent office on 2006-08-03 for surface layer affinity-chromatography.
Invention is credited to Frederick John Rowell.
Application Number | 20060172434 10/542883 |
Document ID | / |
Family ID | 9951423 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172434 |
Kind Code |
A1 |
Rowell; Frederick John |
August 3, 2006 |
Surface layer affinity-chromatography
Abstract
There is described an affinity-chromatography assay system
comprising with an immobilised component containing a bio-reagent
and a flowable component containing a complimentary bio-reagent
characterised in that the immobilised component is supported on a
dip strip or planar surface and the flowable component is adapted
to flow down the dip strip of high density. There is also described
a method of conducting an affinity-chromatography assay which
comprises the use of such an assay system.
Inventors: |
Rowell; Frederick John;
(Durham, GB) |
Correspondence
Address: |
KATTEN MUCHIN ROSENMAN LLP
525 WEST MONROE STREET
CHICAGO
IL
60661-3693
US
|
Family ID: |
9951423 |
Appl. No.: |
10/542883 |
Filed: |
January 20, 2004 |
PCT Filed: |
January 20, 2004 |
PCT NO: |
PCT/GB04/00200 |
371 Date: |
December 27, 2005 |
Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/558
20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 33/558 20060101
G01N033/558 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 20, 2003 |
GB |
0301225.9 |
Claims
1. An affinity-chromatography assay system comprising with an
immobilised component containing a bio-reagent and a flowable
component containing a complimentary bio-reagent characterised in
that the immobilised component is supported on a dip strip or
planar surface and the flowable component is adapted to flow down
the dip strip of high density.
2. An affinity-chromatography assay system according to claim 1
characterised in that the flowable component is of a higher density
than the bulk solution.
3. An affinity-chromatography assay system according to claim 1
characterised in that immunoreagent is an antigen or antibody.
4. An affinity-chromatography assay system according to claim 1
characterised in that the flowable component is retained in a
discrete volume.
5. An affinity-chromatography assay system according to claim 1
characterised in that the constituents of the flowable phase
include a bio-polymer, a detergent and a buffer of optimal pH
6. An affinity-chromatography assay system according to claim 1
characterised in that the immobilised component possesses
properties that result in attraction of the flowable component.
7. An affinity-chromatography assay system according to claim 6
characterised in that the attraction of the flowable component is
achieved by a membrane.
8. An affinity-chromatography assay system according to claim 7
characterised in that the membrane is both hydrophobic and
wettable.
9. An affinity-chromatography assay system according to claim 3
characterised in that the assay is either a competitive or
non-competitive immunoassay using appropriate combinations of
labelled antigen or labelled antibody with their complementary
unlabelled counterparts.
10. An affinity-chromatography assay system according to claim 9
characterised in that the label is a fluorescent or coloured
label.
11. A method of conducting an affinity-chromatography assay which
comprises the use of an assay system according to claim 1.
12. A method according to claim 11 characterised in that the
dipstrip that is stood substantially upright in a buffer
solution.
13. A method according to claim 11 characterised in that the
flowable component is dispensed adjacent the upper or lower part of
the dipstrip.
14. A method according to claim 11 characterised in that the method
comprises the separation of analyte mixtures.
15. A method according to claim 11 characterised in that the
components have different binding affinities for the surface.
16. A method according to claim 11 characterised in that the method
comprises a single step assay.
17. A method according to claim 11 characterised in that the method
comprises the separation of biological polymers.
18. A method according to claim 17 characterised in that the
biological polymers are selected from proteins and DNA/RNA.
19. An affinity-chromatography assay system or a method
substantially as described with reference to the accompanying
examples.
Description
[0001] Immuno-chromatography is currently performed in two major
formats. The first is performed within gels when development is
achieved by passive diffusion or is electrochemically induced, and
the second is performed in flow. In the former using gels, radial
immunodiffusion is the most commonly used system with a preformed
gel often located within a circular plate in which a central well
is present in the gel together with additional wells located around
the edge of the gel. Antiserum or antigen is placed in the central
well and antigen or antiserum is placed in the peripheral wells.
Passive diffusion occurs within the gel and white bands of
immunocomplex are seen within the gel due to antibody-antigen
complex formation. In the electrochemical system antibody/antigen
migration within the gel is induced using an electric current.
[0002] In the flow-based systems the antibody or antigen is often
immobilised within a cartridge which is paced with a liquid flow
system such as a flow injection analysis system. The complementary
antigen or antibody is injected and flows through the cartridge
where specific interactions take place. Often the component that is
injected carries a label that can be detected downstream, thereby
producing a signal. Alternatively flow occurs over a planar surface
as in lateral flow diffusion immunoassay systems where flow is
induced by membrane wetting/capillarity.
[0003] In the new format the equivalent immunoreactions take place
in flow between an immobilised component and one in solution as
above but this now occurs on the surface of a dip strip residing
within a buffer solution. In this case flow across the surface of
the strip occurs due to the higher density of a solution containing
one of the immunoreagents that is initially present at the top of
the strip that is itself standing in the buffer solution. Since the
strip is nearly upright this denser solution slowly rolls down the
surface of the strip presenting the reagent in the flowing phase to
the immobilised reagent on the surface of the dip strip.
[0004] Thus according to the invention we provide an
affinity-chromatography assay system comprising with an immobilised
component containing a bio-reagent and a flowable component
containing a complimentary bio-reagent characterised in that the
immobilised component is supported on a dip strip or other planar
surface and the flowable component of high density is adapted to
flow down the dip strip.
[0005] In order for this phenomenon to work certain criteria must
be met. Firstly the denser solution must be retained in a discrete
volume as it passes as a layer over the surface rather than rapidly
diffusing into the bulk of the buffer solution. Secondly the dip
strip must possess certain properties that result in attraction of
the rolling surface layer again leading to retention of the
integrity of this mobile phase. To achieve the first criterion we
have carefully chosen the constituents of the rolling phase to
include a polymeric agent such as a protein and/or a
polysaccharide, a detergent and a buffer of optimal pH, and for the
second we use a membrane that is both hydrophobic and wettable.
[0006] The system can be used as an immuno-chromatography system
and assays performed in either competitive or non-competitive
immunoassay formats. In the former the immobilised spot is either
antibody or antigen. For immobilised antibody, a labelled antigen
is deposited in a band above the spot in a cellulose square. A drop
of sample containing the antigen is added to this square and after
a suitable interval (1-10 min) the whole strip is immersed in a
buffer solution. The dense mixture of labelled and unlabelled
antigen flows over the spot of antibody and competition for binding
takes place. If the antigen is immobilised at the spot, a labelled
antibody replaces the labelled antigen in the cellulose square and
the assay proceeds as before.
[0007] The system can also be performed in a non-competitive
immunoassay format when a spot of capture antibody is immobilised
on the strip. Labelled antibody is deposited on the cellulose
square above the first spot. The strip is placed in the sample
solution containing the antigen so that the upper square is
immersed. Incubation now takes place during which antigen in the
solution is captured by immobilised antibody on the spot. At the
same time the labelled antibody is reconstituted in a dense
solution that flows over the spot after about 5-10 minutes
following insertion of the strip into the sample solution. This
time lag enables antigen molecules to be captured on the spot prior
to arrival of the surface layer containing the labelled antibody.
This second antibody labels the captured antigen on the spot's
surface.
[0008] In addition any label can be used. If a fluorescent or
coloured label is used with the antibody or antigen, then a
fluorescent or coloured spot will result following the first
incubation, making the assay a single step system.
[0009] If an enzyme label is used then a modified sequence of steps
can be used in the non-competitive assay. In this, enzyme-labelled
antibody is now added to the cellulose square attached to the
bottom of the strip beneath the spot of immobilised capture
antibody. A second cellulose square is also attached as before
above this spot but this contains a dried solution of substrate for
the enzyme plus a biopolymer such as dextran. The strip is placed
in a limited volume of sample as before so as not to wet the upper
cellulose square. The dense reconstituted solution of labelled
antibody flows to the bottom of the container and stays there as a
separate layer. At the same time antigen in solution is captured by
the antibody on the spot. At the end of this incubation step (5-10
minutes) the solution is stirred using the dip strip. This causes
the labelled antibody to be homogeneously distributed within the
solution and the antibody can now bind to the captured antigen on
the spot. Finally the volume in the container is increased to wet
the upper cellulose square. The substrate is now reconstituted as a
dense solution that flows over the spot when substrate to product
conversion takes place resulting in a coloured spot.
[0010] According to a further aspect of the invention we provide a
method of conducting immuno-chromatography assays which comprise
the use of an assay system as hereinbefore described.
[0011] This new surface layer chromatography phenomenon could, in
theory, also be used as a generic chromatographic method for
separation of analyte mixtures if the components have different
binding affinities for the surface. For example, it is well known
that biological polymers such as proteins and DNA/RNA bind to
cellulose nitrate as this is used in DNA- and protein-blotting
following electrophoresis. We have also observed that for
antibodies the rate of binding to this surface is pH sensitive [1].
Hence it should be possible to introduce a mixture of biological
polymers onto a cellulose strip above a cellulose nitrate square.
The pH and density of the buffer used would be such that surface
layer chromatography will ensue when the strip is immersed into a
second buffer solution chosen to optimise the binding of the
biopolymers to the surface. Under these conditions, different
binding interactions will take place between the bio-molecules and
the surface as the mobile phase layer rolls over the surface. This
should lead to separation of the components during this development
phase. The strip would then be removed and the membrane treated to
visualise, using established methods, the now immobilised
components of the mixture.
[0012] The invention will now be illustrated with reference to the
accompanying example.
EXAMPLE 1
[0013] In an example of this new immunochromatographic system that
we term Surface Layer Immuno-Chromatography (SLIC), a small square
of cellulose nitrate membrane is pre-treated via established
methods with a specific antibody to an antigen such as savinase, to
produce a spot of immobilised reagent. This square is stuck on the
surface of a plastic strip pre-coated with one sticky surface.
Above this is stuck a second strip of cellulose impregnated with a
dried solution of the same antibody labelled with the reporter
enzyme alkaline phosphatase, together with bovine serum albumin
(BSA) and Tween 20. The solution used for this deposition is Tris
(pH 9.3) and a volume of 10 .mu.l is used. The composition of this
solution is 0.1% w/v BSA, 0.1% w/v Tween 20, enzyme-labelled
antibody diluted 1:1000 in Tris buffer (0.1M). The reagents in this
format when stored at room temperature in a desiccator are stable
on the strip for at least 14 days.
[0014] To perform the assay, a solution of savinase (0.3 ml) in
Tris buffer is placed in a test tube such as a conventional 96 well
microtitre plate. The dip strip is now inserted into the well when
both squares are covered by the sample. On wetting, the reagents
within the square of cellulose pass into solution. Due to its
higher density this solution now flows as a layer down the surface
of the strip and eventually passes over the lower square containing
the spot of immobilised antibody. In the time interval between
immersion of the strip and arrival of the flowing phase, savinase
molecules in the bulk solution will have been captured by the
immobilised antibodies on the lower spot. On arrival of the flowing
phase, labelled antibody will bind to the captured antigen
molecules as in a conventional sandwich-type ELISA, as the solution
flows over the spot. This first incubation period is typically 15
minutes.
[0015] The strip is removed from the well and placed in wells
containing the commonly used substrate mixture for alkaline
phosphatase, bromochloroindolyl phosphate (BCIP) and nitroblue
tetrazolium salt (NBT). A purple/blue colour develops after 1
minute's incubation, the intensity of which is directly
proportional to the amount of savinase captured on the spot during
the first incubation step, and hence the concentration of savinase
in the sample. The top square is also coloured purple/blue. A scan
of the resulting strips is shown for this analyte. It should be
noted that we use 12 such strips in a comb-like format as this
enables analysis of up to 96 samples/standards (8.times.12) to be
performed with a single microtitre plate. It will be noted that
visual discrimination between the zero and the 5 ng/ml standard is
clearly seen.
EXAMPLE 2
[0016] As in example 1, a small square of cellulose nitrate
membrane with a spot of specific antibody is attached to the
surface of a plastic strip. Below this a second strip of cellulose
(cut as 0.4.times.0.6 cm squares from fast hardened filter paper)
is placed. This square is impregnated with a 5 .mu.l of a diluted
solution of the same antibody labelled with the reporter enzyme
alkaline phosphatase, in a solution of blue dextran, (3% w/v),
dextran (25 w/v), all in Tris buffer with azide, pH 9.3 (containing
0.1% Tween 20 and 0.15 (w/v) bovine serum albumin). The applied
solution is allowed to dry at ambient temperature for 30 minutes. A
third square of fast hardened filter paper is stuck above the first
square and is impregnated with the substrate for alkaline
phosphatase. This is prepared by addition of 15 .mu.l of a mixture
of 1% (w/v) blue dextran, 1% (w/v) dextran and BCIP-NBT stock
solution. It is allowed to dry for 60 minutes at ambient
temperature at which point the square is stuck to the dip
strip.
[0017] The pre-prepared dip strips are then added to microcuvettes
containing 0.6 ml of standards or samples. After 10 minutes a blue
layer of solution is observed at the base of the cuvette. The
strips are used to stir the contents of the cuvettes. This produces
a uniform blue coloration throughout the solution. Incubate for a
further 5 minutes then add 500 .mu.l of substrate buffer solution
to now cover the top square on the dip strip. Incubate for a final
10 minutes, then remove the strips and wash with water.
[0018] When rabbit anti-savinase antibodies are used as the capture
and capping antibodies in the presence of savinase standards, the
following strips are observed when the above protocol is
followed.
[0019] The resulting dip strips are illustrated in FIG. 1, in
which:
[0020] A=zero standards, B=10 ng/ml standards of savinase
* * * * *